Circulating Circular RNAs As Biomarkers For Acute Ischemic Stroke And Its Related Mechanism Research

Aims: Acute ischemic stroke(AIS)is a common acute cerebrovascular disease.Its high disability,lethality,and recurrence rate make it a long-term danger to human health.Therefore,understanding and preventing acute ischemic stroke is of great significance to the world,especially China.Currently,there are no valuable blood-based biomarkers that could be used for diagnosing acute ischemic stroke(AIS)and predicting stroke outcomes.Circ RNAs are stable in peripheral blood owing to their closed ring structure and their participation in various pathophysiological processes associated with stroke,showing their promise as stroke biomarkers.The aim of the present study was to explore circulating circ RNAs associated with AIS,their utility as early diagnostic markers and their significances in predicting stroke outcomes.Furtherly,the present study aimed at exploring the underlying mechanism of circ RNAs and their potential value as treatment target for AISMethods: A circ RNA microarray was conducted to identify differentially expressed circulating circ RNAs in a discovery cohort.Validation and replication were performed in independent cohorts by quantitative real-time polymerase chain reaction(q PCR).Based on the calculated copy number of circ RNAs per microliter plasma,their ROC curve for AIS diagnosis was obtained to analyze their significance as diagnostic biomarker.In addition,pearson correlation analysis was used to analyze the relationship between circ RNAs expression levels and stroke severity in patients with AIS.Additionally,ROC curve for prognosis of AIS patients was analyzed based on circ RNAs expression levels.Platelets,lymphocytes and granulocytes were separated from blood by gradient centrifugation to examine the origins of circ RNAs.Bioinformatics analysis was conducted to uncover the underlying mechanisms of circ RNAs after stroke.The expression level of circ CDC14 A in peri-infarct cortex and plasma of transient middle cerebral artery occlusion(t MCAO)mice were dectected by q PCR.The localization of circ CDC14 A in peripheral blood cells and peri-infarct cortex of t MCAO mice were explored by in situ hybridization and immunofluorescence colocalization staining.In addition,triphenyltetrazolium chloride(TTC)staining were used to detect cerebral infarct volume in t MCAO model mice.Moreover,modified neurological severity score(m NSS)was used to assess neurological defection.Finally,lateral ventricular brain microinjection and tail vein injection of lentivirus were applied to interfere with the expression of circ CDC14 A,thus their effects on behavior,morphology,and molecular biology of t MCAO mice were analyzed.Results: We identified,validated,and replicated three differentially expressed circ RNAs,which were upregulated in patients with AIS compared with HCs(circ FUNDC1: P=0.00014;circ PDS5B: P=4.13*10-9;circ CDC14A: P=1.86*10-9).With an area under the curve(AUC)of 0.875 corresponding to a specificity of 91% and a sensitivity of 71.5%,the combination index of these 3 circ RNAs had diagnostic power for stroke.Among AIS patients,expression levels of these three circ RNAs were positively correlated with infarct volume(circ FUNDC1: P=0.0345,R2=0.02747;circ PDS5B: P=0.04,R2=0.02594;circ CDC14A: P=0.006,R2=0.04591),while no significant correlation was found between circ RNA levels and neurological severity scores.The baseline circ RNA levels showed poor significance,but the change rate of circ RNA levels within the first 7 days of treatment showed significance in predicting stroke outcomes(AUCs of circ FUNDC1,circ PDS5 B,circ CDC14 A and the overall circ RNA set were 0.884,0.953,0.943 and 0.960,respectively).Compared to HCs,circ PDS5 B levels in lymphocytes and granulocytes of AIS patients increased(P<0.01 and P<0.001 respectively),while circ CDC14 A levels only increased in granulocytes(P<0.001).Moreover,the expression levels of circ CDC14 A in plasma and peri-infarct cortex of t MCAO mice were significantly increased,and circ CDC14 A was mainly localized in neutrophils peripherally while in astrocytes in peri-infarct cortex centrally.Tail vein injection of lentivirus to interfere with the expression of circ CDC14 A significantly reduced the infarct volume(P<0.01)and m NSS(P<0.0001)of the t MCAO mice,and improved survival rate within 7 days after modeling(P<0.001).Tail vein injection of si-circ CDC14 A lentivirus significantly reduced the density of activated astrocytes in peri-infarct cortex at 3 days after t MCAO modeling(P<0.0001).Moreover,morphology analysis showed the volume and surface area of activated astrocytes significantly decreased(P<0.0001).Conclusion: A set of circulating circ RNAs—circ FUNDC1,circ PDS5 B and circ CDC14A—could not only serve as biomarkers for AIS diagnosis but also be applied in predicting stroke outcomes.Peripheral interference of circ CDC14 A expression is expected to inhibit ischmic damage by regulating the activation of astrocytes after stroke,which might be a potential therapeutic target for AIS patients.